1. Field
The invention relates to methods of illuminating a specimen in light microscopy. In particular it relates to methods of illumination in various modes, such as bright-field, phase contrast, dark-field, and fluorescence microscopy.
2. Prior Art
LED light sources for microscopes have become plentiful, but very few have been integrated into condensers. Several original shortcomings, such as uncontrolled spectral distribution and low intensity have been overcome, and it is now possible to assume that nearly any classical source of microscope illumination, arc, filament, halogen etc. lamps are replaceable by LEDs or combinations of LEDs.
Three major methods of illumination for microscopes are critical illumination, diffuse illumination, and Köhler illumination. In critical illumination, an image of the light source is focused by a condensing lens on the specimen plane, mainly to maximize brightness. In diffuse illumination, the specimen is illuminated by light that has passed through, for example, a translucent layer that eliminates images, with or without a condenser lens, at the specimen plane. In Köhler illumination, a combination of lenses, typically four, is used to defocus the image of the light source and provide intense uniform, focused illumination at the specimen plane.
Köhler illumination is a method of specimen illumination used for transmitted and reflected light (trans- and epi-illuminated) optical microscopy. Köhler illumination acts to generate an extremely even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp filament) is not visible in the resulting image. Köhler illumination is the predominant technique for sample illumination in modern scientific light microscopy. Köhler illumination, by definition, provides a uniform light field in the specimen plane by focusing the source at the back focal plane of the objective lens. In old and/or simple microscopes a single condenser lens projects an image of the illuminator lamp, often a helical filament, on the specimen plane, thereby producing a confounded combined image of both the specimen and the lamp. Classically, this condition is called critical illumination. In 1893 August Köhler designed a method of illumination which produced a perfectly defocused image of the light source at the sample plane.
A typical light path in a modern microscope includes two iris diaphragms, or apertures, positioned so that one controls the size of the field illuminated and the other controls the amount of light reaching the specimen plane. The operator has numerous adjustments to consider: centering the light source, axially positioning the light source, controlling light source intensity, positioning the condenser (condensing lenses) between the light source and the specimen, centering the condenser, focusing the condenser on the specimen plane (Abbe condenser), and adjusting two apertures for brightness and uniformity of brightness across the specimen plane (Köhler illumination). The application of LEDs to microscope illumination facilitates the elimination of several of these adjustments.